Genome-wide identification of PAR domain protein 1 (PDP1) targets through ChIP-seq reveals the regulation of diapause-specific characteristics in Culex pipiens.

Insects use seasonal diapause as an alternative strategy to endure adverse seasons. This developmental trajectory is induced by environmental cues like short-day lengths in late summer and early fall, but how insects measure day length is unknown. The circadian clock has been implicated in regulating photoperiodic or seasonal responses in many insects, including the Northern house mosquito, Culex pipiens, which enters adult diapause. To investigate the potential control of diapause by circadian control, we employed ChIP-sequencing to identify the downstream targets of a circadian transcription factor, PAR domain protein 1 (PDP1), that contribute to the hallmark features of diapause. We identified the nearest genes in a 10 kb region of the anticipated PDP1 binding sites, listed prospective targets and searched for PDP1-specific binding sites. By examining the functional relevance to diapause-specific behaviours and modifications such as metabolic pathways, lifespan extension, cell cycle regulation and stress tolerance, eight genes were selected as targets and validated using ChIP-qPCR. In addition, qRT-PCR demonstrated that the mRNA abundance of PDP1 targets increased in the heads of diapausing females during the middle of the scotophase (ZT17) compared with the early photophase (ZT1), in agreement with the peak and trough of PDP1 abundance. Thus, our investigation uncovered the mechanism by which PDP1 might generate a diapause phenotype in insects.

The diapausing mosquito Culex pipiens exhibits reduced levels of H3K27me2 in the fat body.

Culex pipiens, the northern house mosquito, is a major vector of West Nile virus. To survive the severe winter, adult mosquitoes enter a diapause programme. Extended lifespan and an increase in lipid storage are key indicators of diapause. Post-translational modifications to histone proteins impact the expression of genes and have been linked to the lifespan and energy utilisation of numerous insects. Here, we investigated the potential contribution of epigenetic alterations in initiating diapause in this mosquito species. Multiple sequence alignment of H3 sequences from other insect species demonstrates a high conservation of the H3 histone in Cx. pipiens throughout evolution. We then compared the levels of histone methylation in the ovaries and fat body tissues of diapausing and non-diapausing Cx. pipiens using western blots. Our data indicate that histone methylation levels in the ovaries of Cx. pipiens do not change during diapause. In contrast, H3K27me2 levels decrease more than twofold in the fat body of diapausing mosquitoes relative to non-diapausing counterparts. H3K27 methylation plays a crucial role in chromosome activation and inactivation during development in many insect species. This is predominantly governed by polycomb repressor complex 2. Intriguingly, a previous ChIP-seq study demonstrated that the transcription factor FOXO (Forkhead box O) targets the genes that comprise this complex. In addition, H3K27me2 exhibits dynamic abundance throughout the diapause programme in Cx. pipiens, suggesting its potential role in the initial activation of the diapause programme. This study expands our understanding of the relationship between alterations in epigenetic regulation and diapause.

Suppressed expression of oxidoreductin-like protein, Oxidor, increases follicle degeneration and decreases survival during the overwintering diapause of the mosquito Culex pipiens.

Throughout diapause in mosquitoes, stress resistance and subsequent prolonged lifespan are a few important features of diapause that are crucial for overwintering success. In the mosquito Culex pipiens, we suggest that oxidoreductin-like protein is involved with these diapause characteristics for overwintering survival. Expression of oxidor was more than two-fold higher in early stage diapausing females compared to their non-diapausing counterparts. Suppression of the gene that encodes oxidoreductin-like protein by RNAi significantly increased the proportion of degenerating follicles in early-stage adult diapausing females. Inhibition of oxidor also significantly reduced the survivability of diapausing females which indicates that this protein plays a key role in protecting multiple tissues during early diapause.

Urogonimus turdi (Digenea: Leucochloridiidae) from the White's Thrush, Zoothera aurea, in the Republic of Korea.

Avian trematodes, Urogonimus turdi (Digenea: Leucochloridiidae), were collected from the intestine of wild birds, Zoothera aurea, 2013-2017 in the Daejeon Metropolitan City, Korea. The body was ellipsoidal, attenuated and/or round ends, 1,987-2,120 long and 819-831 µm wide. The oral sucker was subterminal, rounded anteriorly, and 308- 425×351-432 µm in size; the prepharynx and esophagus were almost lacking; pharynx was well-developed, 142- 179×78-170 µm in size; intestine narrow, bifurcating just after pharynx, ascending to the oral sucker before looping posteriorly and terminating near the posterior end; ventral sucker larger, in almost median, 536-673×447-605 µm and approximately 1.5 times larger than oral sucker. A phylogenetic tree constructed with 18S ribosomal RNA showed inter- and intraspecific relationships. Based on these morphological and molecular findings, we report here a U. turdi from White's thrushes in Korea.

Identification of FOXO targets that generate diverse features of the diapause phenotype in the mosquito Culex pipiens.

Insulin and juvenile hormone signaling direct entry of the mosquito Culex pipiens into its overwintering adult diapause, and these two critical signaling pathways appear to do so by converging on the regulation of forkhead transcription factor (FOXO). Diapause is a complex phenotype, and FOXO emerges as a prime candidate for activating many of the diverse physiological pathways that generate the diapause phenotype. Here, we used ChIP sequencing to identify direct targets of FOXO. The nearest gene in a 10-kb region surrounding a predicted binding site was extracted for each binding site, resulting in a dataset containing genes potentially regulated by FOXO. By selecting candidate genes based on their functional relevance to diapause, we identified five gene categories of potential interest, including stress tolerance, metabolic pathways, lifespan extension, cell cycle and growth regulation, and circadian rhythms. Twelve targets were prioritized for further analysis, 10 of which were validated by ChIP-quantitative PCR and quantitative real-time PCR. These 10 genes activated by FOXO are highly up-regulated during diapause and are thus strong candidates for implementation of the diapause syndrome.

Detection and Identification of Sarcocystis cruzi (Protozoa: Apicomplexa) by Molecular and Ultrastructural Studies in Naturally Infected Korean Cattle (Bos taurus coreanae) from Daejeon, Korea.

To survey the prevalence of Sarcocystis infections, 210 heart samples were collected from Korean native cattle (Bos taurus coreanae) at an abattoir in Daejeon Metropolitan City, Republic of Korea. Sarcocysts were detected form 31 specimens (14.8%) and identified as Sarcocystis cruzi via transmission electron microscopy. The wall of S. cruzi has flattened protrusions that did not contain fibrils or microfilaments. The protrusions arose irregularly from the base, contained a fine granular substance, lacked internal microfilaments, and measured approximately 0.21-1.25 µm in length and 0.05-0.07 µm in width. Sequence analysis revealed 99.5% homology to S. cruzi. This is the first report on the prevalence of S. cruzi in native cattle from the Republic of Korea.

Inter-population variation for Wolbachia induced reproductive incompatibility in the haplodiploid mite Tetranychus urticae.

Recent studies have revealed diverse patterns of cytoplasmic incompatibility (CI) induced by Wolbachia in the two spotted spider mite (Tetranychus urticae Koch). The mechanism of CI consists of two steps: modification (mod) of sperm of infected males and the rescue (resc) of these chromosomes by Wolbachia in the egg, which results in female embryonic mortality (FM), male development (MD) or no CI. Our study reports that Wolbachia infections were highly prevalent infecting all T. urticae populations from various crops in 14 commercial greenhouses in Korea, with two Wolbachia strains expressing distinctive phenotypic effects on hosts. Analyses for wsp gene sequences obtained from collected mite populations revealed all sequences were categorized into two groups (group W1 and W2) discriminated by three diagnostic nucleotides while all Wolbachia strains belonged to the subgroup Ori in Wolbachia supergroup B. Host plants of each mite population were also generally correlated this grouping. Various mating experiments with two mite populations from each group showed that CI patterns and host plants of the mite populations were completely matched with the grouping; no CI (mod(-)resc(+)) for group W1 and mixed pattern of FM and MD (mod(+)resc(+)) for group W2. No distinct changes in fecundity or sex ratio due to Wolbachia infections were observed in four mite populations regardless of Wolbachia grouping. Our study suggests a potential correlation between phenotypic effect of Wolbachia infection and its genetic diversity associated with host plants in Korean mite populations.

A Horsehair Worm, Gordius sp. (Nematomorpha: Gordiida), Passed in a Canine Feces.

Nematomorpha, horsehair or Gordian worms, include about 300 freshwater species in 22 genera (Gordiida) and 5 marine species in 1 marine genus (Nectonema). They are parasitic in arthropods during their juvenile stage. In the present study, the used gordian worm was found in the feces of a dog (5-month old, male) in July 2014. Following the worm analysis using light and scanning electron microscopes, the morphological classification was re-evaluated with molecular analysis. The worm was determined to be a male worm having a bi-lobed tail and had male gonads in cross sections. It was identified as Gordius sp. (Nematomorpha: Gordiidae) based on the characteristic morphologies of cross sections and areole on the cuticle. DNA analysis on 18S rRNA partial sequence arrangements was also carried out, and the gordiid worm was assumed to be close to the genus Gordius based on a phylogenic tree analysis.

Consuming royal jelly alters several phenotypes associated with overwintering dormancy in mosquitoes.

Introduction: Females of the Northern house mosquito, Culex pipiens, enter an overwintering dormancy, or diapause, in response to short day lengths and low environmental temperatures that is characterized by small egg follicles and high starvation resistance. During diapause, Culex pipiens Major Royal Jelly Protein 1 ortholog (CpMRJP1) is upregulated in females of Cx. pipiens. This protein is highly abundant in royal jelly, a substance produced by honey bees (Apis mellifera), that is fed to future queens throughout larval development and induces the queen phenotype (e.g., high reproductive activity and longer lifespan). However, the role of CpMRJP1 in Cx. pipiens is unknown. Methods: We first conducted a phylogenetic analysis to determine how the sequence of CpMRJP1 compares with other species. We then investigated how supplementing the diets of both diapausing and nondiapausing females of Cx. pipiens with royal jelly affects egg follicle length, fat content, protein content, starvation resistance, and metabolic profile. Results: We found that feeding royal jelly to females reared in long-day, diapause-averting conditions significantly reduced the egg follicle lengths and switched their metabolic profiles to be similar to diapausing females. In contrast, feeding royal jelly to females reared in short-day, diapause-inducing conditions significantly reduced lifespan and switched their metabolic profile to be similar nondiapausing mosquitoes. Moreover, RNAi directed against CpMRJPI significantly increased egg follicle length of short-day reared females, suggesting that these females averted diapause. Discussion: Taken together, our data show that consuming royal jelly reverses several key seasonal phenotypes of Cx. pipiens and that these responses are likely mediated in part by CpMRJP1.

Transcription profiling reveals tissue-specific metabolic pathways in the fat body and ovary of the diapausing mosquito Culex pipiens.

The northern house mosquito, Culex pipiens, employs diapause as an essential survival strategy during winter, inducing important phenotypic changes such as enhanced stress tolerance, lipid accumulation, and extended longevity. During diapause, the cessation of reproductive development represents another distinctive phenotypic change, underlining the need for adjusted modulation of gene expressions within the ovary. Although considerable advancements in screening gene expression profiles in diapausing and non-diapausing mosquitoes, there remains a gap in tissue-specific transcriptomic profiling that could elucidate the complicated formation of diverse diapause features in Cx. pipiens. Here, we filled this gap by utilizing RNA sequencing, providing a detailed examination of gene expression patterns in the fat body and ovary during diapause compared to non-diapause conditions. Functional annotation of upregulated genes identified associations with carbohydrate metabolism, stress tolerance, immunity, and epigenetic regulation. The validation of candidate genes using quantitative real-time PCR verified the differentially expressed genes identified in diapausing mosquitoes. Our findings contribute novel insights into potential regulators during diapause in Cx. pipiens, thereby opening possible avenues for developing innovative vector control strategies.

Juvenile hormone III suppresses forkhead of transcription factor in the fat body and reduces fat accumulation in the diapausing mosquito, Culex pipiens.

Juvenile hormone (JH) controls diverse physiological and developmental events including diapause and nutrient metabolism. The focal point of endocrine regulation in adult reproductive diapause is initiated by a halt of JH synthesis. In diapausing females of the mosquito Culex pipiens, the other key molecular event is the signalling pathway from insulin to forkhead of transcription factor (FOXO). We hypothesized that a halt of JH synthesis is related to activation of FOXO, which results in increasing lipid reserves in the fat body at the onset of the diapause programme. In this study, the full-length sequence of the foxo gene in C. pipiens was characterized, and the protein abundance pattern of the foxo gene product was analyzed by immunoblotting and immunohistochemistry. FOXO was much more abundant in the fat body of diapausing females than in the fat body of nondiapausing females; much lower levels were present in other adult tissues. When we topically applied JH III to diapause-destined females, FOXO was suppressed, and fat accumulation was reduced, suggesting an interaction between JH synthesis and FOXO that is critical for expression of the diapause phenotype.

Identification of CYCLE targets that contribute diverse features of circadian rhythms in the mosquito Culex pipiens.

Culex pipiens demonstrates robust circadian rhythms in adult eclosion, flight activity, mating, and development. These rhythmic patterns are believed to be controlled by the endogenous light-entrainable circadian clock that consists of positive and negative regulators working in a transcription-translation feedback loop. Moreover, these mosquitoes undergo seasonal diapause in exposure to the short photoperiod of late summer or early fall. However, the exact genetic and cellular mechanism behind the clock gene-mediated activity pattern, seasonal time measurement, and subsequent diapause initiation still need to be unraveled. To determine the possible linkage between clock genes and downstream processes, here we employed ChIP-sequencing to identify the direct targets of one of the core clock proteins, Cycle (CYC). The nearest genes with peaks mapping to their 1Kb upstream region of the transcription start site were extracted and scanned for consensus E box sequences, resulting in a dataset comprising the target genes possibly regulated by CYC. Based on the highest fold enrichment and functional relevance, we identified genes relating to five gene categories of potential interest, including peptide/receptors, neurotransmission, olfaction, immunity, and reproductive growth. Of these, we validated fourteen genes with ChIP-qPCR and qRT-PCR. These genes showed a significantly high expression in dusk compared to dawn in concert with the activity level of the CYC transcription factor and are thus strong candidates for mediating circadian rhythmicity and possibly regulating seasonal shifts in mosquito reproductive activity.

Suppression of the gene encoding PDZ domain-containing protein decreases cold tolerance and overwintering survival of the mosquito, Culex pipiens (Culicidae: Diptera).

In diapausing mosquitoes, cold tolerance and prolonged lifespan are important features that are crucial for overwintering success. In the mosquito Culex pipiens, we suggest that PDZ domain-containing protein (PDZ) (post synaptic density protein [PSD95], drosophila disc large tumor suppressor [Dlg1], and zonula occludens-1 protein [zo-1]) domain-containing protein is involved with these diapause features for overwintering survival in Culex mosquitoes. The expression level of pdz was significantly higher in diapausing adult females in the early stage in comparison to their nondiapausing counterparts. Suppression of the gene that encodes PDZ by RNA interference significantly decreased actin accumulation in the midgut of early-stage adult diapausing females. Inhibition of pdz also significantly reduced the survivability of diapausing females which indicates that this protein could play a key role in preserving the midgut tissues during early diapause.

Crystallization-based upcycling of iron oxyhydroxide for efficient arsenic capture in contaminated soils.

Arsenic (As)-contaminated soil inevitably exists in nature and has become a global challenge for a sustainable future. Current processes for As capture using natural and structurally engineered nanomaterials are neither scientifically nor economically viable. Here, we established a feasible strategy to enhance As-capture efficiency and ecosystem health by structurally reorganizing iron oxyhydroxide, a natural As stabilizer. We propose crystallization to reorganize FeOOH-acetate nanoplatelets (r-FAN), which is universal for either scalable chemical synthesis or reproduction from natural iron oxyhydroxide phases. The r-FAN with wide interlayer spacing immobilizes As species through a synergistic mechanism of electrostatic intercalation and surface chemisorption. The r-FAN rehabilitates the ecological fitness of As-contaminated artificial and mine soils, as manifested by the integrated bioassay results of collembolan and plants. Our findings will serve as a cornerstone for crystallization-based material engineering for sustainable environmental applications and for understanding the interactions between soil, nanoparticles, and contaminants.

Species-specific environmental DNA analysis of the index species in soil ecosystem, Allonychiurus kimi (Collembola: Onychiuridae).

Collembola are abundant and have significant roles in the soil ecosystem. Therefore, the phenotypic endpoints of Collembola population or community have been used as an effective bioindicator for assessing soil quality. Since the identification and counting the collembolans in the soil is a laborious and costly procedure, environmental DNA (eDNA)-based biomonitoring was proposed as an analysis tool of collembolan species found in the soil. In this study, standard primer sets for the species-specific eDNA analysis using Allonychiurus kimi, a soil bioindicator species was selected. Then, the primers were tested for specificity and sensitivity from the soil samples. Two different eDNA samples were tested: (1) eDNA samples were extracted from the soil with A. kimi individuals (intra-organismal eDNA). (2) The samples from the soil without A. kimi individuals (extra-organismal eDNA). The two primers were confirmed in their sensitivity and specificity to the two types of eDNA samples selected. C t-values from both intra- and extra-organismal eDNA showed the significant correlations to the number of inoculated A. kimi (adj. R 2 = 0.7453-0.9489). These results suggest that in excretion, egg, and other exuviae had a significant effect on eDNA analysis from soil samples taken. Furthermore, our results suggest that environmental factors should be considered when analyzing eDNA collected from soil.

Insulin signaling and FOXO regulate the overwintering diapause of the mosquito Culex pipiens.

The short day lengths of late summer program the mosquito Culex pipiens to enter a reproductive diapause characterized by an arrest in ovarian development and the sequestration of huge fat reserves. We suggest that insulin signaling and FOXO (forkhead transcription factor), a downstream molecule in the insulin signaling pathway, mediate the diapause response. When we used RNAi to knock down expression of the insulin receptor in nondiapausing mosquitoes (those reared under long day lengths) the primary follicles were arrested in a stage comparable to diapause. The mosquitoes could be rescued from this developmental arrest with an application of juvenile hormone, an endocrine trigger known to terminate diapause in this species. When dsRNA directed against FOXO was injected into mosquitoes programmed for diapause (reared under short day lengths) fat storage was dramatically reduced and the mosquito's lifespan was shortened, results suggesting that a shutdown of insulin signaling prompts activation of the downstream gene FOXO, leading to the diapause phenotype. Thus, the results are consistent with a role for insulin signaling in the short-day response that ultimately leads to a cessation of juvenile hormone production. The similarity of this response to that observed in the diapause of Drosophila melanogaster and in dauer formation of Caenorhabditis elegans suggests a conserved mechanism regulating dormancy in insects and nematodes.

Transcript Assembly and Quantification by RNA-Seq Reveals Significant Differences in Gene Expression and Genetic Variants in Mosquitoes of the Culex pipiens (Diptera: Culicidae) Complex.

The taxonomy of Culex pipiens complex of mosquitoes is still debated, but in North America it is generally regarded to include Culex pipiens pipiens, Culex pipiens molestus, and Culex quinquefasciatus (or Culex pipiens quinquefasciatus). Although these mosquitoes have very similar morphometry, they each have unique life strategies specifically adapted to their ecological niche. Differences include the capability for overwintering diapause, bloodmeal preference, mating behaviors, and reliance on blood meals to produce eggs. Here, we used RNA-seq transcriptome analysis to investigate the differential gene expression and nucleotide polymorphisms that may link to the divergent traits specifically between Cx. pipiens pipiens and Cx. pipiens molestus.

Suppression of glycogen synthase expression reduces glycogen and lipid storage during mosquito overwintering diapause.

During diapause in mosquitoes, efficient storage and utilization of energy are crucial for surviving prolonged periods of developmental arrest and for maximizing reproductive success once diapause is terminated and development recommences. In Culex pipiens, glycogen rapidly accumulates during early diapause (7-10 days after adult eclosion) and it is used to maintain energy homeostasis during the first month of diapause. In this study, a gene encoding glycogen synthase, which converts glucose residues into a polymeric chain for storage as glycogen, was characterized. After dsi-RNA directed against glycogen synthase was injected into mosquitoes programmed for diapause (reared under short day lengths), Cx. pipiens were fed 1% d-[13C6]glucose, and the knockdown effects after 7-days were monitored by measuring 13C-labeled carbohydrate accumulation using solid-state NMR. The use of 13C cross-polarization magic-angle spinning spectrum showed a 46% reduction of 13C-labeled glycogen and a 6% reduction in lipid accumulation in glycogen synthase knockdown adult females. In addition, the suppression of glycogen synthase dramatically increased the mortality rate of diapausing Cx. pipiens by 88% at 30-days post injection. These findings indicated that glycogen synthase plays a critical role in regulating glycogen and lipid storages during overwintering diapause, and its function is essential for successful overwintering and survival of Cx. pipiens.

Dsi-RNA knockdown of genes regulated by Foxo reduces glycogen and lipid accumulations in diapausing Culex pipiens.

Culex pipiens is a major carrier of the West Nile Virus, the leading cause of mosquito-borne disease in the continental United States. Cx. pipiens survive overwinter through diapause which is an important survival strategy that is under the control of insulin signaling and Foxo by regulating energy metabolism. Three homologous candidate genes, glycogen synthase (glys), atp-binding cassette transporter (atp), and low-density lipoprotein receptor chaperone (ldlr), that are under the regulation of Foxo transcription factor were identified in Cx. pipiens. To validate the gene functions, each candidate gene was silenced by injecting the target dsi-RNA to female Cx. pipiens during the early phase of diapause. The dsi-RNA injected diapause-destined female post-adult eclosion were fed for 7 days with 10% glucose containing 1% D-[13C6]glucose. The effects of dsi-RNA knockdown on glucose metabolism in intact mosquitoes were monitored using 13C solid-state NMR and ATR-FTIR. Our finding shows that the dsi-RNA knockdown of all three candidate genes suppressed glycogen and lipid biosyntheses resulting in inhibition of long-term carbon energy storage in diapausing females.

Transcription profiling and regulation of fat metabolism genes in diapausing adults of the mosquito Culex pipiens.

Culex pipiens, the mosquito that vectors West Nile virus in North America, overwinters in an adult diapause (dormancy) that is programmed by the short day length and low temperatures of autumn. In response to these environmental signals, females cease feeding on blood and instead seek sources of nectar used to generate the huge lipid reserves required for winter survival. To identify regulatory networks that regulate fat accumulation and fat consumption during diapause, we compared expression of fat-related genes from nondiapausing females with expression of those same genes in early and late diapause and at diapause termination. Among the 31 genes we examined, 4 were expressed more highly in early diapause than in nondiapause, while 14 genes were downregulated in early diapause. In the transition from early to late diapause, 19 genes related to fat metabolism were upregulated. As reported previously, fatty acid synthase, identified as fas-1 in this study, was upregulated in early diapause. Numerous fat metabolism genes, including multiple kinetic classes and genes involved in beta-oxidation, an energy-generation step, were suppressed in early diapause but were highly expressed in late diapause and at diapause termination. RNA interference (RNAi) analysis revealed that the fas-1 gene and others (fas-3 and fabp) have important roles in fat storage during early diapause. When expression of these genes is suppressed, female mosquitoes fail to sequester the lipids needed for overwintering.